PROJECT SUMMARY/ABSTRACT Osteosarcoma (OS) is the most common bone cancer in children and young adults, and outcome for patients with recurrent/refractory disease remains poor. To improve outcome for these patients, we have developed T cells expressing chimeric antigen receptors (CARs) specific for HER2, which is expressed in OS. While HER2- CAR T cells were safe in early phase clinical testing, their anti-OS activity was limited. Lack of efficacy is most likely multifactorial, including limited access to nutrients with the tumor microenvironment (TME). Arginine as emerged as a key amino acid that is critical for T cell function. Indeed, depletion of arginine within the TME has emerged as an immune evasion strategy of tumors. T cells only express the low capacity arginine transporter CAT-1 in contrast to other cells within the TME, which express the high capacity transporter CAT-2b. This differential expression results in arginine depletion away from T cells and thus suppression. To give T cells a competitive advantage in an arginine-depleted environment, we now propose to express CAT-2a in T cells, which has the highest capacity to transport arginine in mammalian cells. I hypothesize that genetically modifying HER2- CAR T cells to express CAT-2a will improve their effector functions within the TME, resulting in enhanced antitumor activity. I will first investigate the functional changes caused by increased arginine import into HER2- CAR.CAT T cells compared to HER2-CAR T cells by analyzing metabolic and memory phenotype, expansion, cytokine production, and tumor killing when challenged with tumor cells or recombinant protein. In addition, I will compare the effector function of HER2-CAR and HER2-CAR.CAT T cells in preclinical models of osteosarcoma. These studies will for the first time evaluate a genetic approach to enable CAR T cells to overcome arginine depletion in the TME, and ultimately could be readily adapted to a broad range of CAR T cell products for pediatric and adult solid tumors. In addition, this proposal will build upon my doctoral immunology training by enabling me to learn genetic engineering, human tumor immunology, advanced metabolic techniques, and preclinical models of cancer immunotherapy in state-of-the-art facilities in Dr. Stephen Gottschalk?s laboratory at St. Jude Children?s Research Hospital.